The effect of culture density on several of the properties of cultured cells

Abstract

A sucrose gradient technique was developed for the preparation of plasma membrane material from SV40 3T3, Py3T3 and HeLa cells. A modification of this method yielded purified nuclei. The plasma membrane material, which was enriched in both 5'-nucleotidase and (Na+K)-ATP ase activities, was used to establish some of the properties of the latter in 3T3 and SV40 3T3 cells. Many of these properties were similar in both cell lines. The effect of conditioning of the growth medium, on the transport of 86Rb into 3T3 and SV40 3T3 cells was investigated. It was shown that conditioning reduced the uptake from growth media and that when the cells were then transferred to Krebs solution, the uptake was apparently increased. The effect of cell population density on several of the properties of 3T3, SV40 3T3, Py3T3 and HeLa cells was examined. When transformed 3T3 cells were grown in medium containing 10% calf serum, the activities of the cell surface enzymes, (Na+K)-ATPase and 5'-nucleotidase, decreased differently with increasing density. The (Na+K)-ATPase activity decreased sharply as the density exceeded 5 x 10<super>4</super> cells/cm2 while the 5'-nucleotidase activity decreased gradually as the density increased. The activity of the latter plateaued so that the overall decrease in the activities of both enzymes was similar. When 3T3 cells were grown under the same conditions, the activities of the cell surface enzymes increased with cell population density. The cell surface enzymes of HeLa cells were shown to decrease with density in a manner similar to those of the transformed 3T3 cells but the decrease in the (Na+K)-ATPase did not exactly follow that of the specific ouabain binding. The effect of culture density on the intracellular enzyme was shown to depend on the cell type as well as the particular enzyme. In all of the 3T3 cell lines, the acid phosphatase activity was observed to increase with increasing density whereas in HeLa cells this enzyme did not alter. In the transformed 3T3 cell lines, the monoamine oxidase activity did not alter with density and the rotenone insensitive NADH-ferricyanide reductase activity increased but in 3T3 cells, the former increased and the latter decreased. In foetal calf serum, the cell surface enzymes were observed to alter differently with respect to cell population density. The cell protein tended to decrease as did the cell volume but these measurements were not directly related. The specific Concanavalin A binding also decreased with cell population density but the decreases were not related to the volume changes. It was concluded that the activity of many of the enzymes of cultured cells is dependent both on the cell population density and the medium in which the cells are grown.